WO2020138503A1 - Agent thérapeutique et prophylactique pour gliome, marqueur de malignité de tumeur cérébrale, marqueur pronostique de tumeur cérébrale, procédé de détermination de la malignité et du pronostic d'une tumeur cérébrale et anticorps inhibant la prolifération tumorale - Google Patents

Agent thérapeutique et prophylactique pour gliome, marqueur de malignité de tumeur cérébrale, marqueur pronostique de tumeur cérébrale, procédé de détermination de la malignité et du pronostic d'une tumeur cérébrale et anticorps inhibant la prolifération tumorale Download PDF

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WO2020138503A1
WO2020138503A1 PCT/JP2019/051635 JP2019051635W WO2020138503A1 WO 2020138503 A1 WO2020138503 A1 WO 2020138503A1 JP 2019051635 W JP2019051635 W JP 2019051635W WO 2020138503 A1 WO2020138503 A1 WO 2020138503A1
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amino acid
hvem
seq
acid sequence
sequence represented
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宮園　浩平
諒 田邉
真大 森川
ヘルディン、カール－ヘンリック
ヴェステルマルク、ベンクト
耕治 玉田
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国立大学法人 東京大学
国立大学法人山口大学
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Priority to JP2020562556A priority Critical patent/JP7457331B2/ja
Priority to US17/419,154 priority patent/US20220105122A1/en
Priority to EP19906351.2A priority patent/EP3936149A4/fr
Priority to CN201980092527.2A priority patent/CN113597315A/zh
Publication of WO2020138503A1 publication Critical patent/WO2020138503A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a therapeutic or preventive agent for glioma.
  • the present invention also relates to a marker for malignancy of brain tumor and a marker for prognosis of brain tumor, and a method of judging malignancy of brain tumor and a method of judging prognosis.
  • the present invention also relates to antibodies that suppress tumor growth.
  • the frequency of occurrence of primary brain tumors in Japan is estimated to be approximately 20,000 per year (prevalence in 2010 is 130.8 per 100,000 persons), and so-called five cerebral tumors are glioma and marrow.
  • Membranoma, pituitary adenoma, schwannoma and craniopharyngioma are known.
  • Resection of the tumor by surgery is generally used for the treatment of brain tumors, but in the case of malignant brain tumors, multidisciplinary treatment combined with an anticancer agent, radiation treatment, etc. is performed.
  • the therapeutic effect depends on how much the tumor can be removed in the first operation. In the case of malignant tumor, if 95 to 98% or more of all tumors are not removed, half of the tumor is removed and the survival prognosis is improved. It is reported that there is not much difference.
  • malignant brain tumors among brain tumors have a high probability of migrating to brain tumors of higher malignancy.
  • Glioblastoma multiforme (hereinafter sometimes referred to as “GBM”) is the most common and most malignant form of malignant brain tumor in adults. Despite advances in surgery, radiation and chemotherapy, glioblastoma patients still have a poor prognosis with median survival times of less than 15 months. Glioblastomas are classified into Proneural, Neural, Classical and Mesenchymal subtypes based on genomic abnormalities from The Cancer Genome Atlas (TCGA) dataset. The highly proliferative nature of glioblastoma cells is due to alterations in several signaling pathways, including oncogenes and tumor suppressor genes.
  • BMP Bone morphogenetic proteins
  • the present inventors have shown that, in addition to BMP-4 inducing the growth arrest and differentiation of glioblastoma stem cells (GIC), the expression of HVEM in specific glioblastoma cells causes BMP. It was found to be suppressed by -4. The present inventors also found that HVEM expression was preferentially increased in glioblastoma multiforme among human adult brain tumors, and that among the four subtypes of GBM, HVEM expression was highest in the Mesenchymal subtype. Found.
  • GAC glioblastoma stem cells
  • the present inventors also found that suppression of HVEM expression in Mesenchymal subtype cell culture attenuates cell proliferation and neurosphere formation, and when Mesenchymal subtype cells with suppressed HVEM expression were injected into the mouse skull, It was found that the tumorigenicity is reduced and the survival time of mice is prolonged. We have also found that overexpression of HVEM enhances glioblastoma cell proliferation and neurosphere formation in cell culture, and intracranial injection of the cells shortens mouse survival. The present inventors further demonstrated that intraperitoneal administration of anti-mouse HVEM antibody to mice in which HVEM-expressing mouse glioma GL261 cells were orthotopically transplanted resulted in decreased tumorigenicity and prolonged survival of mice.
  • the present inventors also found that in GBM, the expression of APRIL, which was not reported as a ligand for HVEM, was high, while the expression of a known ligand for HVEM was low.
  • the inventors have also found that suppressing APRIL expression in Mesenchymal subtype cell cultures attenuates cell proliferation and neurosphere formation.
  • the present inventors also found that APRIL transmits a signal to HVEM by co-culturing HVEM-expressing cells and APRIL-expressing cells.
  • an anti-human HVEM antibody produced by Alpaca suppresses the growth of Mesenchymal subtype cells.
  • the present invention is based on these findings.
  • the present invention aims to provide a novel therapeutic or prophylactic agent for glioma.
  • the present invention also aims to provide markers of malignancy of brain tumors and prognostic markers of brain tumors.
  • Another object of the present invention is to provide a method for determining the malignancy of brain tumors and a method for determining the prognosis of brain tumor patients.
  • Still another object of the present invention is to provide a novel antibody that suppresses tumor growth.
  • a therapeutic or prophylactic agent for glioma which comprises an HVEM inhibitor as an active ingredient.
  • the glioma is any of oligodendroglioma, oligoastrocytoma, astrocytoma, and glioblastoma multiforme.
  • the glioblastoma multiforme belongs to any of Proneural subtype, Neural subtype, Classical subtype, and Mesenchymal subtype.
  • the HVEM inhibitor is an antibody or nucleic acid against HVEM.
  • the antibody against HVEM is an antibody that inhibits the binding between HVEM and a ligand.
  • a therapeutic or prophylactic agent according to any one of [1] to [7] above, which is to be administered to a subject whose HVEM expression level exceeds the HVEM expression level of a healthy subject or the HVEM expression level of a normal tissue sample.
  • a marker of malignancy of brain tumor or a prognosis marker of brain tumor which comprises HVEM protein or HVEM gene.
  • a method for determining the malignancy of a brain tumor which comprises the step of measuring the expression level of HVEM in a target biological sample.
  • Complementarity determining regions 1 to 3 (CDR1, CDR2 and CDR3), and the amino acid sequences of CDR1, CDR2 and CDR3 are (i), (ii), (iii), (iv), ( v), (vi) or (vii) which is the immunoglobulin single variable domain according to the above [15]
  • CDR1 comprising the amino acid sequence represented by SEQ ID NO: 36, the amino acid represented by SEQ ID NO: 37 CDR2 containing the sequence and CDR3 containing the amino acid sequence represented by SEQ ID NO:38;
  • the immunoglobulin single variable domain includes a framework region (FR1, FR2, FR3 and FR4), and the amino acid sequences of FR1, FR2, FR3 and FR4 are (viii), (ix), (x ), (xi), (xii), (xiii) or (xiv), FR1 comprising the immunoglobulin variable domain (viii) amino acid sequence represented by SEQ ID NO: 57 according to the above [16], SEQ ID NO: 58 FR2 containing the amino acid sequence represented by, FR3 containing the amino acid sequence represented by SEQ ID NO:59 and FR4 containing the amino acid sequence represented by SEQ ID NO:60; (Ix) FR1 containing the amino acid sequence represented by SEQ ID NO: 61, FR2 containing the amino acid sequence represented by SEQ ID NO: 62, FR3 containing the amino acid sequence represented by SEQ ID NO: 63 and the amino acid represented by SEQ ID NO: 64 FR4 containing the sequence; (X
  • the immunoglobulin variable domain (xv) according to [15] above, which comprises the polypeptide of (xv), (xvi) or (xvii) below, having the amino acid sequence of any of SEQ ID NOs: 29 to 35.
  • (Xvi) a polypeptide comprising an amino acid sequence having 80% or more identity to any of the amino acid sequences of SEQ ID NOs: 29 to 35, and having HVEM binding ability and tumor growth inhibitory ability;
  • the amino acid sequence of any one of SEQ ID NOs: 29 to 35 which contains an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and/or added, and has HVEM binding ability and tumor growth inhibitory ability
  • [20] A polynucleotide encoding the immunoglobulin single variable domain according to any of [15] to [18] above or the antibody or multimer according to [19] above.
  • a pharmaceutical composition comprising the immunoglobulin single variable domain according to any of [15] to [18] above or the antibody or multimer according to [19] above.
  • a method for treating or preventing glioma which comprises the step of administering an effective amount of an HVEM inhibitor to a subject in need thereof.
  • a method for treating glioma which comprises the step of performing the method for determining the malignancy of a brain tumor according to any of [10] to [12] above, and whether the patient has a high-grade brain tumor.
  • a subject determined to be likely to be affected or a subject determined to be, or likely to be suffering from glioma
  • an effective amount of an anticancer agent in particular, a method of treatment comprising the step of administering an HVEM inhibitor.
  • the agents of the above [1], [23] and [24] are referred to as the "agent of the present invention", and the compositions of the above [1], [23] and [24] are In the specification, they may be referred to as “the composition of the present invention”.
  • a novel therapeutic or prophylactic agent for glioma which is a relatively high frequency of occurrence of brain tumors and which is difficult to cure, and a means for determining malignancy of brain tumors and prognosis of brain tumor patients are provided.
  • glioma especially glioblastoma multiforme
  • the present invention is advantageous in that it can provide a novel therapeutic strategy for malignant brain tumors including glioma.
  • FIG. 1 a is a diagram showing the inhibitory effect of BMP signaling in GBM cells in Example 1.
  • FIG. 1b is a diagram showing the inhibitory effect of BMP signaling in GBM cells in Example 1.
  • Sphere formation of GBM cells (U3024MG, U3031MG and U3054MG) was measured in the presence or absence of 30 ng/mL recombinant human BMP-4.
  • FIG. 2 shows HVEM quantitative RT-PCR analysis of GBM cells treated with BMP-4 in Example 2.
  • FIG. 3a is a diagram showing that the expression level of HVEM is elevated in malignant brain tumors in Example 3. Expression levels of HVEM in normal brain and brain tumor tissues in the TCGA dataset (**P ⁇ 0.01, ***P ⁇ 0.001; two-sided Kruskal-Wallis test with Bonferroni correction).
  • FIG. 3b is a diagram showing that the expression level of HVEM in Example 3 was elevated in malignant brain tumors and correlated with the prognosis of brain tumor patients.
  • FIG. 4c is a diagram showing that blocking of HVEM inhibits neurosphere formation of Mesenchymal subtype cells in cell culture in Example 4. Sphere formation of Mesenchymal subtype cells expressing HVEM shRNA or control shRNA.
  • Example 5a also shows that HVEM silencing in Example 5 attenuates the in vivo tumorigenic activity of Mesenchymal subtype cells. Images of Mesenchymal subtype cells expressing shRNA and firefly luciferase in the mouse skull by an in vivo bioluminescence imaging system. The luminescence intensity of the tumor composed of firefly luciferase-expressing GBM cells orthotopically transplanted to the mouse head was observed 15 weeks after the transplantation. For U3054 MG cells, 4 mice each were used in the experiment.
  • FIG. 5b shows that silencing of HVEM attenuates the in vivo tumorigenic activity of Mesenchymal subtype cells in Example 5.
  • FIG. 6b shows that ectopic HVEM promotes neurosphere formation in non-Mesenchymal subtype cells in Example 6. Sphere formation of non-Mesenchymal subtype cells expressing HVEM or EGFP (control).
  • Figure 7a shows that ectopic HVEM enhances in vivo tumorigenic activity of non-Mesenchymal subtype cells in Example 6. Images from an in vivo bioluminescence imaging system of non-Mesenchymal subtype cells (U3047MG) expressing EGFP or HVEM grown in the mouse skull. The luminescence intensity of a tumor consisting of non-Mesenchymal subtype cells expressing firefly luciferase and EGFP or HVEM orthotopically transplanted in the mouse head was observed 7 weeks after the transplantation.
  • Figures 8a and b show that HVEM is required for in vivo progression of mouse glioma cells in Example 7.
  • FIG. 1 Expression level of HVEM in mouse glioma GL261 cell line.
  • B Intracranial growth of GL261 cells expressing HVEM or control shRNA in the brain of C57BL/6J mice. EGFP and shRNA were co-transduced into GL261 cells with lentivirus. Mouse brain tissue was subjected to hematoxylin and eosin (H&E) staining or bioluminescence imaging of EGFP.
  • H&E hematoxylin and eosin
  • mice 8c shows that HVEM is required for in vivo progression of mouse glioma cells in Example 7.
  • the upper figure shows hematoxylin-eosin stained brain tissue 25 days after intracranial orthotopic transplantation of mouse glioma cells GL261.
  • 9a and b are diagrams showing the expression of HVEM ligands in brain tumors in Example 8.
  • (A) Quantitative RT-PCR analysis of APRIL during lentivirus-mediated shRNA APRIL knockdown in Mesenchymal subtype cells (U3054MG). Data are shown as mean ⁇ SD (n 3 biological replicates).
  • FIG. 10d and e are diagrams showing the effect of APRIL inhibition in Example 9.
  • FIG. 10f is a diagram showing APRIL receptor expression in Mesenchymal subtype cells in Example 9.
  • FIG. 11 is a diagram showing that APRIL transmits a signal of HVEM in Example 10.
  • A HEK293T cells expressing HVEM were co-cultured with HEK293T cells expressing soluble ligands (APRIL, LIGHT or SALM5). Data are presented as mean ⁇ SD of NF- ⁇ B relative activity in HEK293T cells expressing HVEM or control vector.
  • FIG. 12 is a diagram showing the effect of HVEM gene knockout (KO) by genome editing in Example 11.
  • B Human HVEM gene knockout inhibits neurosphere formation in Mesenchymal subtype cells in cell culture.
  • FIG. 13 is a diagram showing the amino acid sequence of the variable region of the alpaca-derived VHH antibody using human HVEM protein as an antigen in Example 12.
  • CDR represents the complementarity determining region, and FR represents the framework region.
  • HVEM is an abbreviation for Herpes Virus entry mediator and refers to a type I transmembrane protein belonging to the TNF/NGF receptor superfamily.
  • HVEM is also called TNFRSF14 (tumor necrosis factor (TNF) receptor superfamily member 14), CD270, LIGHTTR or ATAR. That is, “HVEM” in the present invention is synonymous with “HVEM/TNFRSF14”.
  • HVEM is expressed on various tissues and cells including T cells, B cells, natural killer cells, dendritic cells, hematopoietic cells and non-hematopoietic cells (parenchymal cells) (Pasero C et al., Curr Opin Pharmacol. , 12: 478-485 (2012)).
  • Multiple ligands are known to bind to HVEM, including TNF-related cytokines such as LIGHT and LT ⁇ and non-TNF-related cytokines such as BTLA, CD160 and SALM5.
  • the human HVEM gene is based on the nucleotide sequence published in HGNC:11912
  • the mouse HVEM gene is based on the nucleotide sequence published in MGI:2675303.
  • the human HVEM protein is based on the amino acid sequence published in GenBank Accession No. NP_003811.2
  • the mouse HVEM protein is based on the amino acid sequence published in GenBank Accession No. NP_849262.1. ..
  • human HVEM mRNA is based on GenBank Accession No. NM_003820.3
  • mouse HVEM mRNA is based on GenBank Accession No. NM_178931.2.
  • HVEM inhibitor is used to include a substance that inhibits the expression of HVEM and a substance that inhibits the function of HVEM.
  • the substance that inhibits the expression of HVEM include nucleic acids for HVEM (eg, nucleic acids targeting HVEM such as antisense nucleic acid, siRNA, shRNA, microRNA, gRNA, and ribozyme).
  • the substance that inhibits the function of HVEM includes a substance that interacts with HVEM to inhibit the function, a substance that inhibits the binding between HVEM and a ligand, and the like, and examples thereof include an antibody and an aptamer.
  • APRIL is mentioned as a ligand of HVEM.
  • APRIL is an abbreviation for "a life-inducing ligand” and is also called TNFRSF13 (tumor necrosis factor (TNF) receptor superfamily member 13). That is, “APRIL” in the present invention is synonymous with “APRIL/TNFRSF13”.
  • An antisense nucleic acid is a nucleic acid complementary to a target sequence.
  • the antisense nucleic acid inhibits transcription initiation by triplex formation, suppresses transcription by hybridizing with a site where an open loop structure is locally formed by RNA polymerase, inhibits transcription by hybridizing with RNA that is being synthesized, Inhibition of splicing by hybridization at the junction of intron and exon, suppression of splicing by hybridization with spliceosome formation site, suppression of translocation from nucleus to cytoplasm by hybridization with mRNA, capping site and poly(A) addition site
  • splicing suppression by hybridization with the translation initiation factor translation initiation suppression by hybridization with the translation initiation factor binding site, translation suppression by hybridization with the ribosome binding site near the initiation codon, by hybridization with the mRNA translation region or polysome binding site
  • the expression of the target gene can be suppressed by blocking the elongation of the peptide chain, suppressing the gene expression by
  • the antisense nucleic acid for HVEM is, for example, a single-stranded nucleic acid complementary to a part of the base sequence selected from the aforementioned HVEM gene sequence, the aforementioned HVEM amino acid sequence-encoding nucleotide sequence, and the aforementioned HVEM mRNA sequence.
  • the nucleic acid may be a naturally occurring nucleic acid or an artificial nucleic acid, and may be based on either DNA or RNA.
  • the length of the antisense nucleic acid is usually about 15 bases to about the same length as the entire length of mRNA, preferably about 15 to about 30 bases.
  • the complementarity of the antisense nucleic acid does not necessarily have to be 100%, and may be such that it can bind complementarily to the DNA or RNA encoding HVEM in vivo.
  • siRNA small interfering RNA
  • RISC RNA-induced silencing complex
  • siRNA is obtained by synthesizing a sense strand and an antisense strand oligonucleotide with an automatic DNA/RNA synthesizer, and denaturing them in an appropriate annealing buffer at 90 to 95°C for about 1 minute, and then at 30 to 70°C. It can be prepared by annealing for about 1 to 8 hours.
  • the length of siRNA is preferably 19 to 27 base pairs, more preferably 21 to 25 base pairs or 21 to 23 base pairs.
  • SiRNA against HVEM can be designed based on its nucleotide sequence so as to cause degradation (RNA interference) of mRNA transcribed from the HVEM gene.
  • Examples of siRNA that inhibits the expression of HVEM include siRNA that uses the above-mentioned HVEM mRNA sequence as a target sequence.
  • ShRNA short hairpin RNA
  • shRNA is an artificially synthesized hairpin-type RNA sequence used for gene silencing by RNA interference (degradation of mRNA).
  • shRNA may be introduced into cells by a vector and expressed by U6 promoter or H1 promoter, or an oligonucleotide having an shRNA sequence may be synthesized by a DNA/RNA automatic synthesizer and self-annealed by the same method as siRNA. It may be prepared by The hairpin structure of shRNA introduced into cells is cleaved into siRNA and bound to RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA, whereby sequence-specific gene expression can be suppressed.
  • RISC RNA-induced silencing complex
  • ShRNA for HVEM can be designed based on its nucleotide sequence so as to cause degradation (RNA interference) of mRNA transcribed from the HVEM gene.
  • shRNA that inhibits the expression of HVEM include shRNA whose target sequence is the above-mentioned HVEM mRNA sequence.
  • MiRNA is a functional nucleic acid that is encoded on the genome and undergoes a multistep generation process to finally become a microRNA of about 20 bases. miRNAs are classified into functional ncRNAs (non-coding RNAs, non-coding RNAs: a general term for RNAs that are not translated into proteins), and play an important role in the life phenomenon of regulating the expression of other genes. There is.
  • the expression of the HVEM gene can be suppressed by introducing miRNA having a specific nucleotide sequence into a cell by a vector and administering it to a living body.
  • GRNA guide RNA
  • gRNA specifically recognizes a target sequence, guides binding of Cas9 protein to the target sequence, and enables gene knockout or knockin.
  • the expression of HVEM gene can be suppressed in vivo by administering gRNA targeting HVEM gene in vivo.
  • gRNA is meant to include sgRNA (single guide RNA).
  • the design method of gRNA in the genome editing technology is widely known, and can be appropriately designed by referring to, for example, Benchmarking CRISPR on-target sgRNA design, Yan et.al., Brief Bioinform, 15 Feb 2017.
  • Ribozymes are RNAs that have catalytic activity. Although there are various ribozymes having various activities, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for the purpose of site-specific cleavage of RNA.
  • the ribozyme may have a size of 400 nucleotides or more such as group I intron type and M1RNA contained in RNaseP, and may have about 40 nucleotides called hammerhead type and hairpin type.
  • Aptamers include nucleic acid aptamers and peptide aptamers.
  • Nucleic acid aptamers and peptide aptamers used in the present invention are SELEX method (Systematic Evolution of Ligands by Exponential enrichment), mRNA display (mRNA display) method, etc. It can be obtained by using an in vitro molecular evolution method in which after formation, selection is performed based on affinity.
  • the antisense nucleic acid, siRNA, shRNA, miRNA, ribozyme and nucleic acid aptamer may contain various chemical modifications in order to improve stability and activity.
  • the phosphate residue may be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, and the like.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate and the like.
  • at least a part may be composed of a nucleic acid analog such as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the antibody against HVEM is an antibody that specifically binds to HVEM and that inhibits the function of HVEM by binding.
  • the antibody against HVEM the immunoglobulin single variable domain of the present invention described below, the antibody of the present invention and the immunoglobulin single variable domain multimer of the present invention can be used.
  • An antibody against HVEM can be produced according to a known antibody or antiserum production method using HVEM protein or a part thereof as an antigen.
  • the HVEM protein or a part thereof can be prepared by known protein expression methods and purification methods.
  • Examples of the HVEM protein include, but are not limited to, human HVEM and the like defined by the above-mentioned sequence information of HVEM.
  • HVEM proteins from various organisms may be used as the immunogen.
  • Antibodies against HVEM that can be used in the present invention can also be produced via the phage display method (see, for example, FEBS Letter, 441:20-24 (1998)).
  • glioma is a general term for tumors that arise from neuroectodermal tissue of brain parenchyma, and accounts for 30 to 40% of primary intracranial tumors, and is the most frequent brain tumor. .. Gliomas include oligodendroglioma, oligoastrocytoma, astrocytoma, and glioblastoma multiforme. “Glioblastoma multiforme” is also called glioblastoma or atypical glioma and is a glioma mainly composed of undifferentiated cells derived from stellate cells.
  • Glioblastoma multiforme nuclear polymorphism is prominent, and necrosis and vascular endothelial proliferation are observed.
  • Glioblastoma multiforme is fast-growing, extensively infiltrating, and often occurs in the adult cerebrum. It has been reported that glioblastoma multiforme is classified into four subtypes of Proneural, Neural, Classical, and Mesenchymal (Verhaak R G et al., Cancer Cell, 17: 98-110 (2010)).
  • HVEM is highly expressed in genoblastoma of the Mesenchymal subtype
  • the agents and compositions of the present invention have a glioblastoma multiforme (especially of the Mesenchymal subtype).
  • it can be preferably used for reducing the risk of developing glioma (particularly glioblastoma multiforme of the Mesenchymal subtype) as described below.
  • the HVEM inhibitor which is an active ingredient of the present invention, is used for a subject whose HVEM expression level exceeds the HVEM expression level of a healthy subject or the HVEM expression level of a normal tissue sample (for example, a brain tumor patient), particularly a brain tumor patient.
  • a HVEM inhibitor can be administered to a subject that highly expresses HVEM therein.
  • the subject to be treated and prevented according to the present invention may be HVEM expression-dependent glioma or HVEM ligand-dependent glioma. Whether the subject highly expresses HVEM, whether the glioma is HVEM-dependent, and whether the glioma is HVEM-ligand-dependent was performed, for example, on brain tumor patients.
  • the brain tissue excised at the time of brain surgery can be evaluated, and details can be determined according to the determination method of the present invention described below and the procedure described in the examples.
  • the agents and compositions of the present invention can also be administered to subjects at risk of developing glioma (especially glioblastoma multiforme), thereby reducing the risk of developing glioma.
  • a subject at risk of developing glioma is a subject who has no subjective symptoms of glioma, or has not been diagnosed with glioma, but who is at risk of developing glioma in the future. And a brain tumor patient who has not been diagnosed with glioma or a brain tumor patient who has undergone resection of brain tumor tissue.
  • “reducing the risk of developing glioma” means reducing the probability of developing glioma, and reducing the probability of developing glioma improves the prognosis of malignant brain tumors.
  • a composition for reducing the risk of developing glioma and a composition for reducing the risk of developing glioma which comprises an HVEM inhibitor as an active ingredient
  • a prognosis improving agent in the treatment of malignant brain tumor and a composition for a prognosis improving agent in the treatment of malignant brain tumor, which comprises an HVEM inhibitor as an active ingredient.
  • the agent and composition of the present invention can be provided as a medicine or a pharmaceutical composition.
  • the drug and the pharmaceutical composition of the present invention comprise an HVEM inhibitor and a pharmaceutically acceptable carrier.
  • the pharmaceutical products and pharmaceutical compositions of the present invention also include pharmaceutical products and pharmaceutical compositions intended for gene therapy.
  • Such drugs and pharmaceutical compositions contain, as an active ingredient, nucleic acids targeting HVEM such as antisense nucleic acid, siRNA, shRNA, microRNA, gRNA, and ribozyme.
  • the drug and the pharmaceutical composition of the present invention may contain an active ingredient other than the HVEM inhibitor, or may be used in combination with an active ingredient other than the HVEM inhibitor or a drug or a pharmaceutical composition containing the same.
  • the active ingredient other than the HVEM inhibitor include anticancer agents (particularly, anticancer agents for treating malignant brain tumor).
  • the route of administration is not particularly limited as long as a therapeutic or prophylactic effect for glioma (particularly glioblastoma multiforme) can be obtained.
  • Parenteral administration eg, intravenous administration, local administration (including local administration using a catheter), subcutaneous administration, intraperitoneal administration) is preferable.
  • an appropriate dosage form can be selected according to a specific administration form, and examples thereof include injections and suppositories.
  • These preparations can be prepared using a pharmaceutically acceptable carrier by a method usually used in this field (for example, a known method described in the 15th Revised Japanese Pharmacopoeia General Rules for Preparation, etc.).
  • Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. Are listed.
  • the dose of the HVEM inhibitor in the present invention can be determined depending on the type of active ingredient, sex of the subject, age and weight, symptoms, dosage form, administration route, and the like.
  • the dose per adult is, for example, 0.0001 mg to 1000 mg/kg body weight.
  • the range can be determined, but is not limited thereto.
  • the daily dose of an HVEM inhibitor for an adult can be determined depending on the type of active ingredient, sex of the subject, age and weight, symptoms, dosage form, administration route, and the like.
  • the active ingredient may be administered once a day or in 2 to 4 divided doses.
  • the agents and compositions of the present invention include not only humans in need thereof but also mammals other than humans (for example, mouse, rat, rabbit, dog, cat, cow, horse, pig, sheep, goat, monkey). Can also be administered to.
  • HVEM glioblastoma multiforme belonging to Mesenchymal subtype
  • HVEM prognostic marker of brain tumor
  • high expression of HVEM correlates with brain tumor, particularly glioblastoma multiforme belonging to Mesenchymal subtype, and high HVEM. Expression was shown to correlate with poor prognosis in glioblastoma patients. It was also confirmed that overexpression of HVEM promotes the growth of glioblastoma multiforme cells, neurosphere formation, and tumor growth in vivo, respectively. Therefore, according to the present invention, a marker of brain tumor malignancy consisting of HVEM protein or HVEM gene and a prognosis marker of brain tumor consisting of HVEM protein or HVEM gene are provided.
  • the marker for malignancy of brain tumor of the present invention can be used for determining the malignancy of brain tumor in a brain tumor patient, and can also be used for determining glioma (particularly glioblastoma multiforme).
  • the brain tumor prognosis marker of the present invention can be used for predicting or estimating the prognosis of a brain tumor patient in the treatment of brain tumor. Therefore, these markers of the present invention are useful as an index when determining a therapeutic strategy for brain tumors.
  • the HVEM protein or HVEM gene is preferably of human origin.
  • the HVEM gene may be DNA consisting of the genomic sequence of the HVEM gene, or may be mRNA of the HVEM gene or cDNA obtained by reverse transcribing the mRNA of the HVEM gene. ..
  • the marker of the present invention can be implemented according to the method of determining the malignancy of brain tumor of the present invention and the method of determining the prognosis of a brain tumor patient of the present invention described below.
  • a method for determining the malignancy of brain tumor which comprises the step of measuring the HVEM expression level in a biological sample of a subject (particularly a brain tumor patient). Provided.
  • the “biological sample” means a sample separated from a living body, and examples thereof include a brain tissue excised during brain surgery.
  • a step of measuring the expression level of HVEM in a biological sample of a test subject is carried out.
  • the HVEM expression level can be measured in vitro by a known method.
  • the expression level of HVEM in a biological sample can be measured based on the expression level of HVEM protein.
  • the expression level of the HVEM protein can be measured by a known detection means using a specific binding substance for the HVEM protein, for example, ELISA, Western blot, immunohistochemical staining and the like. it can.
  • An antibody is mentioned as a specific binding substance for the HVEM protein, and the agents and compositions of the present invention and those described for the antibody of the present invention can be used.
  • the expression level of HVEM in the biological sample can also be measured based on the expression level of the HVEM gene.
  • the expression level of the HVEM gene can be measured by a known method such as RT-PCR, quantitative RT-PCR, DNA microarray analysis and Northern blotting.
  • the probe and primer set used for measuring the expression level of the HVEM gene can be prepared based on the sequence information of the HVEM gene described above with reference to the examples described later.
  • a step of determining the degree of malignancy of tumor cells contained in a biological sample can be further carried out based on the HVEM expression level measured in the step (A). ..
  • the biological sample when the HVEM expression level in the biological sample of the subject exceeds the HVEM expression level in the biological sample of the healthy subject or in the normal tissue sample (preferably when it exceeds the significant amount), the biological sample Are shown to contain highly aggressive tumor cell populations (eg, glioma cells, especially glioblastoma multiforme cells).
  • the method for determining malignancy of the present invention is (B1) when the HVEM expression level in the target biological sample exceeds the HVEM expression level in the healthy target biological sample or the normal tissue sample (preferably If it exceeds by a significant difference), the biological sample may further comprise the step of determining that the biological sample contains a high-grade tumor cell population.
  • a step of determining malignancy of brain tumor in a subject from which a biological sample is collected can be further performed based on the HVEM expression level measured in the step (A). ..
  • the HVEM expression level in the biological sample of the subject exceeds the HVEM expression level in the biological sample of the healthy subject or in the normal tissue sample (preferably if it exceeds with significant difference)
  • the subject is malignant. It is shown to suffer from frequent brain tumors such as gliomas, especially glioblastoma multiforme.
  • the method for determining malignancy of the present invention is (B2) when the HVEM expression level in the target biological sample exceeds the HVEM expression level in the healthy target biological sample or the normal tissue sample (preferably The method may further comprise the step of determining that the subject suffers from a high-grade brain tumor (if significantly exceeded).
  • a tumor cell population with high malignancy such as glioma cells can be detected in a test biological sample.
  • a highly malignant brain tumor such as glioma can be detected in a subject. Therefore, the method for determining malignancy according to the present invention is useful in providing appropriate information for determining a treatment policy for brain tumor. That is, the method of determining malignancy of the present invention can be used as an auxiliary to the diagnosis and/or differentiation of glioma (particularly glioblastoma multiforme), and whether the subject has glioma. The judgment can be finally made by a doctor, possibly in combination with other findings.
  • a method for determining the prognosis of a brain tumor patient which comprises the step of measuring the HVEM expression level in a biological sample of a subject (particularly a brain tumor patient).
  • the step of measuring the HVEM expression level in the biological sample of the test subject is carried out.
  • the HVEM expression level can be measured in the same manner as the method for determining malignancy of the present invention.
  • the step of determining the prognosis of the subject from which the biological sample is collected can be further performed based on the HVEM expression level measured in the step (C). In this step, if the HVEM expression level in the biological sample of the subject exceeds the HVEM expression level in the biological sample of the healthy subject or in the normal tissue sample (preferably with a significant difference), the prognosis of the subject Is shown to be bad.
  • the method for determining malignancy of the present invention is (D) when the HVEM expression level in the target biological sample exceeds the HVEM expression level in the healthy target biological sample or the normal tissue sample (preferably The method may further include the step of determining that the prognosis of the subject is poor, if the results are significant.
  • the HVEM expression level (cutoff value) in a biological sample of a healthy subject or in a normal tissue sample is a biological sample previously collected from a plurality of healthy subjects, or a plurality of subjects (including a brain tumor patient) in advance.
  • the average value calculated by measuring the HVEM expression level in the normal tissue collected from can be used, or the value calculated by the above formula (1) can be used.
  • poor prognosis means that survival rate is lower within a predetermined period, brain tumor with high malignancy (for example, glioma, particularly glioblastoma multiforme). ) Is likely to develop.
  • the prognosis of a brain tumor patient can be predicted or estimated in the treatment of brain tumor. Therefore, the prognosis determination method of the present invention is useful in that it provides appropriate information for determining the treatment policy for brain tumors. That is, the prognosis determination method of the present invention can be used as an adjunct to the treatment of brain tumors (eg, glioma, particularly glioblastoma multiforme), and the determination of whether or not the subject has a poor prognosis depends on the case. Some can be combined with other findings and eventually done by a doctor.
  • brain tumors eg, glioma, particularly glioblastoma multiforme
  • an immunoglobulin single variable domain that specifically binds to HVEM and suppresses growth of tumor cells (especially growth of malignant tumor cells) is provided.
  • the single variable domain of the invention is capable of binding to HVEM with an EC50 value of less than 80 nM.
  • the single variable domain of the present invention can be characterized by complementarity determining regions 1 to 3 (CDR1, CDR2 and CDR3), and CDR1, CDR2 and CDR3 can be characterized by the aforementioned (i), (ii), (iii), ( iv), (v), (vi) and (vii) can be specified by the combination of CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 complementarity determining regions 1 to 3
  • CDR1, CDR2 and CDR3 can be characterized by the aforementioned (i), (ii), (iii), ( iv), (v), (vi) and (vii) can be specified by the combination of CDR1, CDR2 and CDR3.
  • the single variable domain of the invention can also be characterized by framework regions 1 to 4 (FR1, FR2, FR3 and FR4), wherein FR1, FR2, FR3 and FR4 are described above (viii), (ix), ( x), (xi), (xii), (xiii) and (xiv) can be specified by a combination of FR1, FR2, FR3 and FR4.
  • the single variable domain of the present invention can be specified by the complementarity determining regions 1 to 3 and the framework regions 1 to 4, and in this case, the single variable domain of the present invention is FR1-CDR1- from the N-terminal side. FR2-CDR2-FR3-CDR3-FR4 can be connected in this order.
  • CDR1, CDR2 and CDR3 of the single variable domain of the present invention are the combination of (i) above, (viii) can be selected as the combination of FR1, FR2, FR3 and FR4.
  • the combination of FR1, FR2, FR3 and FR4 can select the above (ix).
  • the combination of FR1, FR2, FR3 and FR4 can select the above (x).
  • CDR1, CDR2 and CDR3 of the single variable domain of the present invention are the combination of (iv) above, the combination of FR1, FR2, FR3 and FR4 can select the above (xi).
  • the combination of FR1, FR2, FR3 and FR4 can select the above (xii).
  • the combination of FR1, FR2, FR3 and FR4 can select the above (xiii).
  • the combination of FR1, FR2, FR3 and FR4 can select the above (xiii).
  • the combination of FR1, FR2, FR3 and FR4 can select the above (xiv).
  • the single variable domain of the present invention can also be specified by the single variable domain sequenced in the Examples (polypeptide of (xv) above), but in the present invention, in addition to this, the above (xv) Polypeptides that are substantially identical to the polypeptides described above are also included in the single variable domain of the present invention.
  • polypeptides substantially identical to the single variable domain of the present invention include humanized single variable domains.
  • Methods for humanizing single variable domains are known, for example, the sequence of a framework region of a naturally occurring amino acid sequence of a single variable domain may be compared to one or more closely related amino acid sequences of a human single variable domain.
  • the potentially useful humanized substitutions were identified by comparison with the corresponding framework sequences of, and the potentially useful humanized substitution(s) thus determined were
  • a humanized single variable domain can be obtained by introducing it into the amino acid sequence of the single variable domain.
  • the humanized single variable domain thus obtained may be subjected to confirmatory tests for affinity to target, stability and other desired properties to determine the amino acid sequence of the appropriate humanized single variable domain. it can.
  • a polypeptide substantially identical to the single variable domain of the present invention can be represented by a polypeptide selected from the above (xvi) and (xvii).
  • identity is used to include “homology”.
  • identity is, for example, the degree of identity when the sequences to be compared are properly aligned (alignment), and means the appearance rate (%) of exact matches of amino acids between the sequences. ..
  • identity for example, the presence of a gap in the sequence and the nature of the amino acid are considered (Wilbur, Natl. Acad. Sci. U.S.A. 80:726-730 (1983)).
  • the alignment can be performed by using an arbitrary algorithm, for example, specifically, BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol.
  • the identity in the above (xvi) can be 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • amino acid sequence in the amino acid sequence, one or more amino acids have been deleted, substituted, inserted and/or added means, for example, the degree of occurrence by a known method such as site mutagenesis. It means that, or a naturally occurring number of amino acids have been modified by deletion, substitution, insertion and/or addition.
  • the number of amino acids modified by the substitution or the like is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or It is one.
  • the modification may occur, for example, continuously or discontinuously.
  • the insertion of the amino acid in the above (xvii) includes, for example, insertion inside the amino acid sequence. Further, the addition of the amino acid in the above (xvii) may be, for example, addition to the N-terminal or C-terminal of the amino acid sequence, or addition to both N-terminal and C-terminal.
  • the amino acid substitution in (xvii) above means that the amino acid residue constituting the amino acid sequence is replaced with another type of amino acid residue.
  • the amino acid substitution in (xvii) above may be, for example, a conservative substitution.
  • Constant substitution means the replacement of one or more amino acids with another amino acid and/or amino acid derivative so as not to substantially alter the function of the protein.
  • the amino acid to be substituted and the amino acid after substitution preferably have similar properties and/or functions, for example.
  • the indices of hydrophobicity and hydrophilicity, chemical properties such as polarity and charge, or physical properties such as secondary structure are similar.
  • amino acids or amino acid derivatives with similar properties and/or functions are known in the art.
  • non-polar amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
  • polar amino acids neutral amino acids
  • examples of polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine and cysteine.
  • examples of the amino acid (basic amino) acid having a positive charge include arginine, histidine and lysine, and examples of the amino acid having a negative charge (acidic amino acid) include aspartic acid and glutamic acid.
  • Preferred amino acid modifications in the above (xvii) include one amino acid substitution, two amino acid substitution, three amino acid substitution, four amino acid substitution, five amino acid substitution, six amino acid substitution or seven amino acids, and more preferably the substitution is conservative. It is a substitution. Preferred amino acid modifications in (xvii) also include those in which the framework regions 1 to 4 are modified, and more preferred are those in which the framework regions 1 to 4 are modified. The preferable amino acid modification in the above (xvii) is also one amino acid substitution, two amino acid substitution, three amino acid substitution, four amino acid substitution, five amino acid substitution, six amino acid substitution or seven amino acids, and the modification is a framework.
  • the modification is one amino acid substitution, two amino acid substitutions, three amino acid substitutions, four amino acid substitutions, five amino acid substitutions, six amino acid substitutions or seven amino acids, and the alterations Occurs only in the framework regions 1 to 4.
  • an antibody and immunoglobulin single variable domain multimer comprising a single variable domain of the invention.
  • Such antibodies include so-called heavy chain antibodies (or VHH antibodies) composed of only two heavy chains as variable regions.
  • the multimer includes a complex in which a plurality of single variable domains of the present invention are bound to each other directly or via a linker, and such a complex includes 1 other than the single variable domain of the present invention.
  • One kind or two or more kinds of optional components for example, physiologically active peptide, stabilizing substance may be further linked.
  • the single variable domain, antibody and multimer of the present invention can be produced, for example, by expressing a polynucleotide encoding these in a host and recovering the expression product. Therefore, according to yet another aspect of the invention, a polynucleotide encoding a single variable domain, antibody or multimer of the invention, and a polynucleotide of the invention or a polynucleotide of the invention is operably linked.
  • a host cell into which the vector has been introduced is provided.
  • the “polynucleotide encoding a single variable domain or the like” can be specified based on the amino acid sequence of the single variable domain or the like based on the genetic code (ie, codon).
  • the "polynucleotide” includes DNA and RNA, and further includes modified forms thereof and artificial nucleic acids, and is preferably DNA.
  • DNA includes cDNA, genomic DNA, and chemically synthesized DNA.
  • the polynucleotide of the present invention is not particularly limited as long as it can be expressed in the host to be used and is composed of codons encoding a single variable domain having HVEM binding activity. Codons may be optimized to allow expression or to increase the expression level. Codon optimization can be performed by a known method usually used in this field.
  • the single variable domain and the like of the present invention can be expressed in and using a variety of host cells, such as bacterial cells, molds, animal cells, plant cells, baculovirus/insect cells or yeast cells. .. Expression vectors for expressing the single variable domain and the like of the present invention are known, and a vector suitable for various host cells can be used.
  • bacterial cells or cultured cells When extracting a single variable domain or the like expressed in a host from cultured bacterial cells or cells, after culturing, the bacterial cells or cultured cells are collected by a known method, suspended in an appropriate buffer solution, and After destroying the bacterial cells or cells by sonication, lysozyme and/or freeze-thawing, a soluble extract is obtained by centrifugation or filtration, and a known separation/purification method is appropriately combined from the obtained extract.
  • a single variable domain of interest can be obtained.
  • Known separation and purification methods include solubility-based methods such as salting-out and solvent precipitation methods, dialysis methods, ultrafiltration methods, gel filtration methods, SDS-PAGE and other methods that mainly utilize differences in molecular weight, and ions.
  • the single variable domain, antibody or multimer of the present invention has HVEM binding activity and can suppress tumor growth.
  • the substance that inhibits the expression of HVEM or the substance that inhibits the function of HVEM can be used for the treatment or prevention of glioma
  • the single variable domain, antibody or multimer of the present invention has a pharmaceutical composition. It can be used as an active ingredient of a product, and particularly as an active ingredient of the agents and compositions of the present invention (ie, therapeutic and prophylactic agents for glioma).
  • a method of treating or preventing glioma comprising administering to a subject in need thereof an effective amount of an HVEM inhibitor.
  • Another aspect of the present invention also provides a method for reducing the risk of developing glioma, comprising administering an effective amount of an HVEM inhibitor to a subject in need thereof.
  • a method for improving prognosis in the treatment of malignant brain tumor which comprises administering an effective amount of an HVEM inhibitor to a subject in need thereof.
  • the method of the present invention can be carried out as described for the agents and compositions of the present invention.
  • the method for determining malignancy of the present invention is carried out, and then an effective amount of an anti-cancer agent (for example, an anti-cancer agent for treating malignant brain tumor, preferably an HVEM inhibitor, particularly HVEM) is used.
  • an anti-cancer agent for example, an anti-cancer agent for treating malignant brain tumor, preferably an HVEM inhibitor, particularly HVEM
  • a subject or a glioma that is determined to have, or is likely to have, a high-grade brain tumor that has a substance that inhibits the binding to APRIL.
  • a method for treating malignant tumor or glioma is provided, which comprises administering to a subject determined to be likely to suffer from the disease.
  • the method of the present invention can be carried out according to the description of the method for determining malignancy of the present invention and the description of the agent and composition of the present invention.
  • an HVEM inhibitor for the manufacture of a therapeutic or prophylactic agent for glioma, or as a therapeutic or prophylactic agent for glioma.
  • an HVEM inhibitor for the production of an agent for reducing the risk of developing glioma, or as an agent for reducing the risk of developing glioma.
  • an HVEM inhibitor for producing a prognosis improving agent in the treatment of malignant brain tumor, or as a prognosis improving agent in the treatment of malignant brain tumor.
  • the use of the present invention can be carried out as described for the agents and compositions of the present invention.
  • an HVEM inhibitor as a therapeutic or prophylactic agent for glioma, for use in the treatment or prevention of glioma, or for use in the therapeutic or prophylactic method of the present invention.
  • HVEM is also used as an agent for reducing the risk of developing glioma, for use in reducing the risk of developing glioma, or for use in the method for reducing risk of the present invention.
  • An inhibitor is provided.
  • an HVEM inhibitor as a prognosis improving agent in the treatment of malignant brain tumor, for use in improving the prognosis in the treatment of malignant brain tumor, or for use in the prognosis improving method of the present invention.
  • the above HVEM inhibitors can be implemented as described for the agents and compositions of this invention.
  • non-therapeutic means not including an act of operating, treating or diagnosing a human (that is, a medical act for a human), specifically, a doctor or a doctor's instruction. It means that the person does not include a method of performing surgery, treatment or diagnosis on a human.
  • Example 1 Inhibitory effect of BMP-4 on proliferation and neurosphere formation of GBM cell line
  • Culture conditions of human glioblastoma cells Human glioblastoma multiforme cells, U3024MG, U3031MG and U3054MG cell lines were humanized.
  • Human glioblastoma cell culture http://www.hgcc.se/#, sometimes referred to herein simply as "HGCC” to obtain glioblastoma stem cells (glioma-initiating cells).
  • GAC Global System for a cell line
  • GAC glioblastoma cell culture
  • B-27 supplement (manufactured by Thermo Fisher Scientific), N-2 supplement (manufactured by Thermo Fisher Scientific), 20 ng/mL epidermal growth factor (EGF, manufactured by PeproTech) ) And basic fibroblast growth factor-basic (bFGF, manufactured by PeproTech) and DMEM/F12 (manufactured by Thermo Fisher Scientific) and Neurobasal medium (manufactured by Thermo Fisher Scientific). ).
  • RNA sequence analysis was performed to identify the BMP target gene using human glioblastoma cell TGS-04 (Raja E. et al., Oncogene. , 36: 4963-4974 (2017)).
  • the expression level of genes controlled by BMP-4 stimulation was analyzed based on the FPKM value (fragments per kilobase of exon per million fragments), and the FPKM value was suppressed to 1/2 or less by BMP-4 stimulation.
  • the genes that were extracted were extracted.
  • the primers used for the quantitative RT-PCR are shown in Table 1.
  • the complementary DNA was synthesized using PrimeScript II 1st strand cDNA synthesis kit (Takara Bio).
  • HVEM H9 hESC-derived human neural stem cells
  • Each prepared shRNA was introduced into the Mesenchymal subtype cells (U3024MG cells, U3031MG cells or U3054MG cells) cultured under the conditions described in Example 1(1). Then, HVEM expression in GBM cells (U3024MG cells, U3031MG cells or U3054MG cells) into which each shRNA was introduced was analyzed by quantitative RT-PCR. Specifically, RT-PCR was performed according to the method described in Example 2(2), using the SEQ ID NOS: 1 to 4 described in Table 1 as the primers.
  • HVEM Inhibition of HVEM by antibody GBM cells (U3024MG cells, U3031MG cells or U3054MG cells) using an antibody against human HVEM (MAB356, manufactured by R&D Systems) or a mouse IgG1 antibody (MAB002, manufactured by R&D Systems) as a control.
  • human HVEM MAB356, manufactured by R&D Systems
  • mouse IgG1 antibody MAB002, manufactured by R&D Systems
  • Example 5 Suppression of in vivo tumor growth of GBM cells by silencing of HVEM (1) Inhibition of HVEM by shRNA in GBM cells Firefly luciferase (Luc2, manufactured by Promega, the same applies hereinafter) was used as a pENTR201 vector (Thermo Fisher Scientific). Same as described in Example 4(1), except that the recombination between pENTR201-Luc2 and CS-CMV-RfA vector was catalyzed by LR clonase (Thermo Fisher Scientific).
  • the firefly luciferase Luc2 was introduced into Mesenchymal subtype cells (U3031 MG cells or U3054 MG cells) to prepare Mesenchymal subtype cells expressing firefly luciferase.
  • Mesenchymal subtype cells U3031 MG cells or U3054 MG cells
  • the control shRNA #1 SEQ ID NO: 5
  • HVEM shRNA #1 or #2 SEQ ID NO: 7 or 8
  • Mesenchymal subtype cells expressing firefly luciferase and control shRNA or HVEM shRNA were prepared.
  • Example 6 Promotion of cell proliferation, neurosphere formation and in vivo tumor growth of GBM cells by overexpression of HVEM
  • HVEM Overexpression of HVEM in vitro Enhanced Green Fluorescent Protein (EGFP), HVEM or Luc2 cDNA is cloned into pENTR201 vector (manufactured by Thermo Fisher Scientific), and recombination between pENTR201 and CSII-EF-RfA or CS-CMV-RfA vector is carried out by LR clonase (manufactured by Thermo Fisher Scientific).
  • EGFP Green Fluorescent Protein
  • HVEM or Luc2 cDNA is cloned into pENTR201 vector (manufactured by Thermo Fisher Scientific)
  • CSII-EF-RfA or CS-CMV-RfA vector is carried out by LR clonase (manufactured by Thermo Fisher Scientific).
  • GBM non-Mesenchymal subtype cells (U3047MG, U3085MG and U3017MG cells) in which EGFP or HVEM were cultured under the conditions described in Example 1(1), except that they were catalyzed by Example 4(1). Was expressed in.
  • Example 7 Inhibition of tumorigenicity of mouse glioma cells by HVEM inhibition (1) HVEM expression and serum HVEM function was analyzed using mouse glioma cell line GL261 (Perkin Elmer). DMEM (manufactured by Thermo Fisher Scientific) medium (differentiation medium containing fetal calf serum) supplemented with 10% fetal calf serum (manufactured by Thermo Fisher Scientific) of GL261 cells or serum-free according to Example 1(1) Cultured in stem cell medium. Quantitative RT-PCR was performed using the primers shown in Table 3 according to the method described in Example 2(2).
  • Example 4 Tumorigenic activity of HVEM
  • a vector having a DNA sequence and an EGFP sequence encoding HVEM shRNA #1 or #2 (SEQ ID NO: 13 or 14) or control shRNA #1 (SEQ ID NO: 5) shown in Table 4 was prepared as Example 4 It was introduced into GL261 cells according to the method described in (1).
  • the GL261 cells were intracranial orthotopically transplanted into C57BL/6J mice to examine the tumorigenic activity of HVEM in the GL261 cells after suppressing the expression of HVEM. Specifically, a total of 1 ⁇ 10 5 viable cells were transplanted at a position 2 mm to the right of the sagittal suture from the previous section of the skull and 3 mm below the surface of the skull.
  • mice transplanted with glioblastoma cells were transcardially perfused with 4% paraformaldehyde.
  • Mouse brain tissues were cryoprotected with 10-20% sucrose, and tissue sections were stained with hematoxylin and eosin.
  • the results were as shown in Figure 8b.
  • GL261 cells treated with control shRNA formed tumors after cell implantation (EGFP signal was observed), whereas mouse glioma cells treated with HVEM shRNA did not form tumors (FIG. 8b). From these results, it was confirmed that the tumorigenic activity of glioma cells can be suppressed by suppressing the expression of HVEM in glioma cells.
  • Hematoxylin-eosin staining in brain tissue confirmed that tumor formation was suppressed in the control group 25 days after GL261 cell transplantation, but tumor formation was suppressed in the group to which the anti-mouse HVEM antibody was administered. All mice in the control antibody treated group died within 35 days, while all mice in the anti-mouse HVEM antibody treated group survived longer than 45 days. From these results, it was confirmed that by inhibiting the function of HVEM in glioma cells, the tumorigenic activity of glioma cells could be suppressed, and the survival period could be extended.
  • Example 8 Expression of HVEM ligands in GBM (1) Expression of HVEM ligands in GBM To investigate the function of HVEM ligands in brain tumors, known ligands for APRIL and HVEM (LIGHT, LTA) were analyzed using the GDC TCGA dataset. , BTLA, CD160, SALM5) mRNA expression levels were analyzed. The results were as shown in Figure 9a. It was observed that APRIL was highly expressed in normal brain tissue and GBM as compared with the known ligand of HVEM.
  • beta DuoSet ELISA (DY211, R&D Systems), human LIGHT/TNFSF14 Quantine ELISA kit (DLIT00, R&D Systems), auxiliary reagent kit 2 (DY008, R&D Systems). Absorbance at 450 nm and 570 nm was measured with a Model 680 microplate reader (Bio-rad) or Enspire (Perkin Elmer). The results were as shown in Figure 9b. The concentrations of LTA and LIGHT in the culture supernatant were below the detection sensitivity. On the other hand, it was confirmed that APRIL was present at a concentration of 50 pg/mL or higher in normal brain tissue (hNSC) and all 13 types of GBM, and that the expression level in GBM was higher than that in normal brain tissue.
  • hNSC normal brain tissue
  • APRIL is a ligand of the TNF superfamily, and BCMA/TNFRSF17 and TACI/TNFRSF13B are known as APRIL receptors. It has not been previously reported that APRIL binds to HVEM and transmits a signal.
  • Example 9 Inhibition of GBM cell proliferation and neurosphere formation by inhibition of APRIL
  • Example 10 Action of APRIL as a HVEM ligand (1) Identification of HVEM ligand A firefly luciferase gene (Luc2, Promega) is located downstream of the minimal promoter having a NF- ⁇ B responsive element, and Renilla is located downstream of the CMV promoter. A luciferase gene (hRluc, Promega) was cloned into pCS-NF- ⁇ B-RE-minP-Luc2-CMV-hRluc, respectively.
  • Human-derived HVEM was introduced into HEK293T cells using Lipofectamine 2000 (manufactured by Thermo Fisher Scientific, the same applies below), and then the NF- ⁇ B-responsive Luc2 gene and the hRluc gene constitutively expressed were introduced ( HVEM expressing cells).
  • human-derived LIGHT, APRIL, and SALM5 genes were cloned into a pENTR4-CMV vector, and recombination between pENTR4-CMV and CS-RfA-EF-PuroR was carried out by LR clonase (Thermo Fisher Scientific). Catalyzed.
  • LIGHT, APRIL, and SALM5 were respectively expressed in HEK293T cells different from those described above using Lipofectamine 2000 to prepare cells expressing soluble ligand (ligand-expressing cells).
  • ligand-expressing cells As a control cell, a cell expressing the NF- ⁇ B reporter gene, but expressing neither HVEM nor ligand was prepared (Empty).
  • HVEM-expressing cells and ligand-expressing cells were co-cultured, and the activity of NF- ⁇ B in the HVEM-expressing cells was evaluated using the Dual luciferase reporter assay kit (Promega, hereinafter the same).
  • Example 11 Knockout effect of HVEM gene using CRISPR/Cas9
  • CRISPR/Cas9 system Lentifectamine 2000 which is a lentiCRISPR v2 vector encoding the gRNAs shown in Table 8 targeting the DNA sequences shown in Table 7, was prepared.
  • HCas9 and each gRNA were introduced into Mesenchymal subtype cells (U3054MG cells) or mouse glioma cell line GL261 (Perkin Elmer). Each cell was cloned by the limiting dilution method.
  • the gRNA sequence was selected with free software (CHOP CHOP: http://chopchop.cbu.uib.no/).
  • Example 1 In vitro cell proliferation assay The cells prepared in (1) above were subjected to an in vitro cell proliferation assay according to the method described in Example 1(3).
  • the GL261 cells were cultured in a serum-free stem cell medium as described in Example 1(1).
  • Sphere formation assay The cells prepared in (1) above were subjected to a sphere formation assay according to the method described in Example 1 (4).
  • Example 12 Preparation of HVEM antibody using alpaca (1) Preparation of antibody in alpaca HEK293T cells expressing human HVEM gene (GenBank Accession No. NM_003820.3 or HGNC:11912) were immunized subcutaneously in the alpaca, and anti-human. HVEM Alpaca VHH antibody was generated. VHH antibodies having the amino acid sequences shown in Table 9 were obtained. Complementarity determining regions (CDRs) were identified according to Kabat's taxonomy. The result was as shown in FIG.
  • the anti-histidine tag antibody anti-His-tag mAb-HRP-DirecT (MBL) labeled with horseradish peroxidase HRP was reacted, and the enzyme reaction between HRP and the substrate was performed using ELISA POD Substrate TMB Kit (Nacalai Tesque). It was measured.
  • Non-linear regression was performed on the sigmoid curve based on the above to determine the 50% effective concentration EC50.
  • the EC50 of VHH#1 to 5 was all less than 80 nM.
  • the VHH#6 and VHH#7 clones were obtained by antibody screening using the phage display method.
  • VHH antibody Mesenchymal subtype cell line (U3054MG cells) was treated with VHH(I) (VHH#3) and VHH(II) (VHH#2) at a concentration of 100 to 300 nM.
  • U3054MG cells were cultured for 7 days under the conditions of 37° C. and 5% CO 2.
  • Cell growth was quantified using Cell Count Reagent SF (Nacalai Tesque) and the cell growth was measured for the PBS-treated group. The result was as shown in FIG. Proliferation was shown to be suppressed in cells treated with VHH.

Abstract

L'objectif de la présente invention est de fournir un nouvel agent thérapeutique ou prophylactique pour un gliome, un marqueur de malignité de tumeur cérébrale, un marqueur de pronostic de tumeur cérébrale, un procédé de détermination de la malignité d'une tumeur cérébrale, et un procédé de détermination du pronostic d'un patient atteint d'une tumeur cérébrale. La présente invention concerne : un agent thérapeutique et prophylactique pour un gliome, ledit agent contenant un inhibiteur du HVEM en tant que principe actif ; un marqueur de malignité de tumeur cérébrale et un marqueur de pronostic de tumeur cérébrale, chaque marqueur comprenant une protéine HVEM ou un gène HVEM ; et un procédé de détermination de la malignité d'une tumeur cérébrale et un procédé de détermination du pronostic d'un patient atteint d'une tumeur cérébrale, chaque procédé comprenant une étape de mesure du taux d'expression de HVEM dans un échantillon biologique collecté à partir d'un sujet.
PCT/JP2019/051635 2018-12-28 2019-12-27 Agent thérapeutique et prophylactique pour gliome, marqueur de malignité de tumeur cérébrale, marqueur pronostique de tumeur cérébrale, procédé de détermination de la malignité et du pronostic d'une tumeur cérébrale et anticorps inhibant la prolifération tumorale WO2020138503A1 (fr)

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JP2020562556A JP7457331B2 (ja) 2018-12-28 2019-12-27 神経膠腫の治療および予防剤、脳腫瘍の悪性度のマーカーおよび脳腫瘍の予後マーカー、脳腫瘍の悪性度および予後の判定方法並びに腫瘍増殖を抑制する抗体
US17/419,154 US20220105122A1 (en) 2018-12-28 2019-12-27 Therapeutic and prophylactic agent for glioma, brain tumor malignancy marker, brain tumor prognostic marker, method for determining malignancy and prognosis of brain tumor and antibody inhibiting tumor proliferation
EP19906351.2A EP3936149A4 (fr) 2018-12-28 2019-12-27 Agent thérapeutique et prophylactique pour gliome, marqueur de malignité de tumeur cérébrale, marqueur pronostique de tumeur cérébrale, procédé de détermination de la malignité et du pronostic d'une tumeur cérébrale et anticorps inhibant la prolifération tumorale
CN201980092527.2A CN113597315A (zh) 2018-12-28 2019-12-27 用于治疗和预防神经胶质瘤的药剂、脑肿瘤恶性程度标志物、脑肿瘤预后标志物、确定脑肿瘤恶性程度和预后的方法以及抑制肿瘤增殖的抗体

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CN113597315A (zh) 2021-11-02
EP3936149A1 (fr) 2022-01-12

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